Addiction is a chronic, relapsing, debilitating condition for which few treatments are available. All addictive substances act by increasing release or impairing reuptake of dopamine (DA) by neurons of the ventral tegmental area (VTA). Understanding the molecular determinants of DA neuron firing rate, and its plasticity in response to drugs of abuse, is therefore crucial to the development of therapies for addiction. G protein gated inward rectifying potassium (GIRK) channels conduct a hyperpolarizing current that reduces DA neuron firing rate, but their expression is reduced 24 hours after exposure to psychostimulants. Our lab has identified sorting nexin 27 (SNX27) as an important positive regulator of GIRK channel expression in vivo. Selective deletion of SNX27 from DA neurons (SNX27DAKO) led to reduced GIRK currents and increased acute locomotor response to cocaine. However, the effect of SNX27 on the pathological learning underlying addiction, as well as the subcellular mechanism and circuit-level specificity of SNX27 regulation of GIRK channels, are unknown. The present proposal addresses these questions using an array of behavioral, electrophysiological and molecular techniques. In Aim 1, the effect of SNX27 on pathological learning underlying addiction will be measured using several behavioral tests including conditioned place preference (CPP) and locomotor sensitization with cocaine, and two-bottle choice with methamphetamine. In Aim 2, the subcellular mechanism of SNX27 regulation of GIRK channels will be addressed. It has been shown that SNX27 regulates other targets via interaction with the retromer complex; therefore, conditional knockout mice lacking the retromer protein VPS26 will be bred from existing floxed lines. These mice will be subjected to electrophysiological and behavioral experiments, and are predicted to exhibit the same phenotypes as SNX27DAKO mice. In Aim 3, the circuit-level specificity of SNX27 regulation of GIRK channels will be explored. The VTA contains multiple distinct subpopulations of DA neurons which have different projection targets. Retrograde labeling with viral constructs will be used to selectively label subpopulations of DA neurons in SNX27DAKO mice, and these neurons will be recorded to determine whether SNX27 regulates GIRK channels in these neuronal populations. These experiments will determine the relevance of SNX27 to addiction, the mechanism by which it regulates GIRK channels, and in which VTA neurons it does so. This will increase our understanding of the changes that occur in the VTA in addiction, paving the way for the development of therapies to treat this challenging disorder.